L-cysteine, PIP3 and Insulin Signaling in Diabetes Abstract Diabetes have become an epidemic and remain a major public health issue worldwide. The primary purpose of this application is to discover the mechanisms by which L-cysteine (LC) supplementation improves glucose homeostasis in diabetic patients. Diabetic patients have lower blood levels of L-cysteine (LC), hydrogen sulfide (H2S), and glutathione (GSH). Supplementation with cysteine-rich proteins (whey protein and ?-lactoalbumin), LC, or N-acetyl cysteine (NAC) has been shown to lower glycemia in diabetic animal studies. However, the molecular mechanism by which LC increases glucose utilization and lowers glycemia is not known. Our study demonstrated activation of PI3K and inhibition of NF-?B in the liver and reduction in blood glucose in LC supplemented ZDF rats, a model of type 2 diabetes. Further studies using an adipocyte cell model showed that LC caused PI3K activation, PTEN inhibition, and an increase in PIP3 (phosphatidylinositol-3,4,5 trisphosphate) and glucose utilization in high glucose (HG)-treated cells. The effect of LC on PIP3 and glucose utilization was prevented by PAG (propargylglycine), an inhibitor of cystathionine-?-lyase (CSE), which catalyzes H2S formation from LC. Treatment with LC, H2S, or PIP3 increased the phosphorylation of IRS1, AKT, and PKC?/?,as well as GLUT4 activation and glucose utilization in HG-treated cells. These studies provide evidence for a novel molecular mechanism by which LC can increase PIP3 and upregulate the metabolic actions of insulin, thus improving glucose metabolism. This application will test the hypothesis that LC upregulates both the insulin dependent (PI3K/AKT/PKC?/?) and insulin independent (SIRT1, AMPK) signaling cascades of glucose metabolism mediated by PIP3 upregulation. The role of H2S and GSH in PIP3 upregulation will be dissected by using specific antisense approaches and specific knock-out mouse models (SIRT KO; CSE KO, which impairs H2S, and GCLM KO, which impairs GSH synthesis). Data will be analyzed statistically. The understanding and validation of the mechanisms by which LC supplementation improves glucose homeostasis and lowers glycemia should support the design of clinical interventions using novel molecules (containing sulfide and cysteine moieties) to improve glucose metabolism and prevent CVD in diabetes. The long-term goal is to discover a relatively low-cost dietary supplement that could be used as an adjuvant therapy for CVD prevention in T2D.